MICROBIOLOGICAL AND PHYSICOCHEMICAL ASSESSMENT - PowerPoint Presentation

1. MICROBIOLOGICAL AND PHYSICOCHEMICAL ASSESSMENT OF ARTISANALLY PRODUCED “ALHEIRA” FERMENTED SAUSAGES IN NORTHERN PORTUGALSara Coelho-Fernandes, Odete Zefanias, Gisela Rodrigues, Ana Sofia Faria, Ângela Fernandes, Lillian Barros, Vasco Cadavez, Ursula Gonzales-Barron1st International Electronic Conference on Food Science and Functional Food

2. Alheira is a traditional non-ready-to-eat sausage produced mainly in Northern PortugalTraditionally made of a mix of poultry and pork, bread and seasoningsNew formulations using game meat, codfish, mushrooms or even vegetarian/vegan options are also available in the marketIntroductionFigure 1. Ingredients used in alheira formulation

3. Alheira productionCooked meats are shredded and mixed with salt, garlic, spices and sliced bread soaked in hot broth to form a non-uniform paste This paste is stuffed into natural casings, and left to dry and mature at cold temperatures for 7-14 daysFigure 2. Alheira production fluxogramFigure 3. Alheira stuffing process

4. Quality of artisanal alheiraQuality characteristics of alheira (physicochemical, nutritional, microbiological and sensorial attributes) are highly variable between regional producers, but also between batches of production of the same enterpriseDifferent or not-sufficiently controlled processing variables and hygiene in the elaboration Different ingredients and their qualityDifferent recipes

5. ObjectivesTo evaluate the variability in relevant physicochemical and microbiological attributes of alheira sausages elaborated by representative artisanal producers of Northern PortugalTo understand the associations between these attributes through the derivation of 3D quality maps based on principal component analysis

6. Trás-os-MontesLisbon •Materials and methodsVila RealBragançaChavesMirandelaMogadouroMiranda do DouroValpaçosPurchased 1-2 days after production and subjected to physicochemical and microbiological analysis within 24 hours after purchaseCasings were carefully removed from the sausages under aseptic conditions, and the contents were divided for physicochemical and microbiological analysesFigure 4. Districts of Portugal. The region of Trás-os-Montes appears in yellow.

7. Materials and methodsPhysicochemical analysispHWater activity (aw)Moisture contentAshes contentProtein contentMicrobiological analysisTotal mesophilesLactic acid bacteria (LAB in MRS and M17)Staphylococcus aureusPresumptive Clostridium perfringens Presence of Salmonella spp.

8. Materials and methodsStatistical analysisData from the 11 attributes were subjected to a principal component analysis (PCA) to summarize the information provided by the physicochemical and microbiological characteristics as well as their interrelationshipsFrom the 3D-PCA, maps of physicochemical and microbiological quality were built from the projection of sample scores onto the span of the principal componentsScores were clustered by artisanal producer (i.e., location; not disclosed in the present study)

9. Results and discussionThe physicochemical and microbiological quality of alheira sausages presented considerable variabilityPhysicochemical analysispH4.034 – 4.606aw 0.9758 – 0.9969Moisture content45.39 – 58.36%Protein content17.78 – 28.00% db Ash content 2.79 – 4.71% dbMicrobiological analysisMesophilic counts7.161 – 9.679 log CFU/g LAB counts7.704 – 11.00 log CFU/gS. aureus counts1.699 – 6.021 log CFU/g Presumptive C. perfringens<0.699 – 1.699 log CFU/gSalmonella spp. was detected in 4 of the 8 sampled artisanal producers at an incidence of 0.20 (one positive sample out of the five samples tested)Tables 1-2. Producer-specific mean values for physicochemical and microbiological analysis

10. Results and discussionThree meaningful components were extracted from the PCA, accounting for 63% of data variabilityVariablePC1PC2PC3CommunalitiespH-0.230.350.861.5aw0.24-0.840.171.2Moisture-0.05-0.73-0.041.0Ashes-0.480.470.052.0Protein0.11-0.200.781.2Total mesophiles0.85-0.14-0.261.2Staphylococcus aureus0.580.470.372.7Clostridium spp.0.250.700.111.3LAB on MRS0.79-0.050.121.1LAB on M170.810.14-0.341.4Salmonella spp.0.350.040.101.2Proportion Variance0.260.210.16-Cumulative Variance0.260.480.63-Table 3. Coefficients of correlation of the physicochemical and microbiological characteristics with the three Varimax-rotated factors (PC1, PC2, PC3) along with communalities and explained variances

11. Results and discussionThe first component (PC1) explained 26% of data variabilityHighly correlated with LAB (on MRS agar (R=0.79); on M17 agar (R=0.81)) and mesophiles (R=0.85) and more weakly correlated with S. aureus (R=0.58)PC1 was labelled longer processing duration, as longer fermentation times (or more efficient fermentations) tend to producer greater populations of mesophiles and LABIf S. aureus contaminates the alheira mixture, its survival depends on an insufficient drop in pH during the first stage of fermentation. It would explain the weaker correlation of S. aureus with PC1, since in some cases this pathogen can either increase or decrease during processing.

12. Results and discussionProducers 1, 6, 7 and 8 seem to employ a longer processing time for the production of alheira sausages, or have a more efficient fermentation processProducer 5 appears to have the shortest alheira production time, or has delayed fermentationFigure 5. Map of the first and second principal component of the physicochemical and microbiological characteristics of alheira sausage with projections of samples from eight artisanal producers of Northern Portugal

13. Results and discussionPC2 (21% of total variability) was highly and inversely correlated with moisture (R=-0.73) and aW (R=-0.84), and directly correlated with presumptive C. perfringens counts (R=0.70)The inverse correlations imply that drier alheiras tended to present higher counts of C. perfringensPC2 was labelled as greater dehydrationGreater dehydration of alheira sausages can arise from longer drying times or higher drying temperatures

14. Results and discussionProducers 1, 6 and 8 produced sausages with overall higher moisture content, yet, of variable moisture (i.e., larger ellipses along PC2 axis)Producers 3, 4 and 5 elaborated drier sausages of more consistent moisture content (i.e., smaller ellipses along PC2)Producer 2 elaborated the most dehydrated sausages, although their drying process may be not fully controlled (i.e., large ellipse along PC2 axis)Figure 5. Map of the first and second principal component of the physicochemical and microbiological characteristics of alheira sausage with projections of samples from eight artisanal producers of Northern Portugal

15. Results and discussionThe third component (PC3) explained 16% of the data variability, and is highly correlated with only two variables, pH (R=0.86) and protein content (R=0.78) Since in alheiras the main source of protein is the meat, PC3 can be labelled as higher meat proportion in the formulationThe probable tendency of artisanal producers to use pig meat of high pH (i.e., DFD meats) may explain why protein content and pH of alheiras seem so highly associatedAnother explanation is that formulations with a lower proportion of meat are compensated with a higher proportion of regional bread, a foodstuff of lower pH. Thus, batters of higher proportion of meat will tend to have higher pH

16. Results and discussionFigure 6. Map of the first and third principal component of the physicochemical and microbiological characteristics of alheira sausage, with projections of samples from eight artisanal producers of Northern Portugal.Producers 1, 4 and 7 employed a higher concentration of meat in their formulations

17. ConclusionsThis work identified three quality axes underpinning the variability in artisanal alheiras: duration of fermentationextent of dehydrationproportion of meat in formulationIt has also highlighted the need to implement better microbiological control and process standardization during the production of artisanal alheiras

18. The authors are grateful to EU PRIMA programme and the Portuguese Foundation for Science and Technology (FCT) for funding the ArtiSaneFood project (PRIMA/0001/2018). The authors are grateful to FCT and FEDER under Programme PT2020 for financial support to CIMO (UIDB/ 00690/2020). Dr. Gonzales-Barron acknowledges the national funding by FCT, P.I., through the Institutional Scientific Employment Programme contractAcknowledgements

19. Thank you for your attention!